The immune system ordinarily functions to direct protective immune responses against microorganisms and other harmful foreign materials. In the context of autoimmune diseases and transplant rejection, however, these normally beneficial immune responses can mediate deleterious and often fatal effects. In the case of autoimmunity, antigens present in the body's own tissues become targets for autoreactive immune responses that cause tissue destruction and other disease symptoms.
Immune responses in mammals are mediated by a diverse array of peripheral blood cells called leukocytes. Leukocytes arise from hematopoietic stem cells which undergo self-renewal and differentiation into two precursor lineages—the myeloid and lymphoid lines. Further differentiation occurs among these lineages to produce monocyte, eosinophil, neutrophil, basophil, megakaryocyte, and erythroid cells from the myeloid line, and T lymphocytes, B lymphocytes, and NK cells from the lymphoid line.
T lymphocytes include CD8+ T cells (cytotoxic/suppressor T cells), and CD4+ T cells distinguished in part by their expression of cell surface molecules, CD8, and CD4, respectively, which function to enhance the avidity with which T cells bind antigen-bearing or target cells, and may also promote the interaction of the TCR with cognate antigen. Bierer et al., Ann. Rev. Immunol. 7:579-99, 1989.
CD4+ T cells play a key regulatory role with respect to other immune system cell types, acting as “T helper” or “T inducer” cells when activated. By virtue of this central regulatory role, CD4+ T cells are key players in the pathogenesis of various autoimmune diseases, including multiple sclerosis (MS), rheumatoid arthritis (RA), diabetes, sarcoidosis, autoimmune uveitis, chronic beryllium disease, and are also considered to play a causal role in transplant rejection and graft-versus-host disease (GVHD) (Swanborg, J. Immunol 130:1503-05, 1983; Cush, Arthritis Rheum. 31: 1230-38, 1988; Caspi, J. Immunol 140:1490-95, 1988; Cobbold et al., Nature 312:54851, 1988; Steinman, Sci. Am. 269:106-14, 1993).
CD4+ T cells mediate their role in autoimmune disease by responding in an antigen-specific manner to “autoantigens” associated with target cells or tissues. Pathogenic CD4+ T cells migrate or “home” to target tissues bearing autoantigen and selectively produce T-helper type 1 (Th1) cytokines, which trigger recruitment and activation of other lymphocytes and monocytes that may destroy target tissues and cause other adverse disease sequelae (Weinberg, et al., J. Immunol 148:2109-17, 1992; Weinberg et al., J. Immunol 152:4712-5721, 1994).
Normal activation of T lymphocytes occurs when the T cells interact with antigen-presenting cells (APCs) bearing cognate antigen (Ag) in the context of a major histocompatibility complex (MHC) protein. The specificity of T cell responses is conferred by a polymorphic, antigen-specific T cell receptor (TCR). T cell activation is mediated by TCR recognition of the Ag presented on the surface of the APC as a processed peptide bound to the MHC molecule.
Two distinct classes of MHC molecules occur in humans and other mammals, termed MHC class I and MHC class II. Both classes of MHC molecules comprise complexes formed by association of multiple polypeptide chains, and each includes a trans-membrane portion that anchor the complex into the APC membrane. MHC class I molecules are comprised of an α-polypeptide chain non-covalently associated with a β2-microglobulin chain. The α-chain of MHC class I includes three distinct domains, termed the α1, α2 and α3 domains. The three-dimensional structure of the α1 and α2 domains of MHC I molecules forms a peptide binding groove (alternatively referred to herein as the peptide binding cleft or pocket) which binds cognate Ag for presentation to T-cells. The α3 domain is an Ig-fold like domain that includes a trans-membrane sequence to anchor the α-chain into the cell membrane of the APC. MHC class I complexes, when associated with antigen in the presence of appropriate co-stimulatory signals, stimulate CD8+ cytotoxic T-cells to kill target cells in an Ag-specific manner.
The genes that encode the various polypeptide chains that associate to form MHC complexes in mammals have been studied and described in extensive detail. In humans, MHC molecules (with the exception of class I β2-microglobulin) are encoded in the HLA region of the genome, located on chromosome 6. There are three class I MHC α-chain-encoding loci, termed HLA-A, HLA-B and HLA-C. In the case of MHC class II proteins, there are three pairs of α and β chain loci, termed HLA-DR(A and B), HLA-DP(A and B), and HLA-DQ(A and B). In rats, the class I α gene is designated RT1.A, while the class II genes are termed RT1.B α and RT1.B β. More detailed description regarding the structure, function and genetics of MHC complexes can be found, for example, in Immunobiology: The Immune System in Health and Disease by Janeway and Travers, Current Biology Ltd./Garland Publishing, Inc. (1997), and in Bodmer et al. (1994) “Nomenclature for factors of the HLA system” Tissue Antigens vol. 44, pages 1-18.
The specificity of T cell responses is conferred by a polymorphic, antigen-specific T cell receptor (TCR). TCRs comprise multi-chain, α/β heterodimeric receptors, which are activated in an Ag-specific manner by Ag processed and presented on the surface of APCs as a peptide bound to the MHC complex. X-ray crystallographic data demonstrate that peptides from processed antigen bind to MHC II proteins in a membrane distal pocket formed by the β1 and α1 domains (Matsui et al., Science 254:1788-91, 1991; Nag et al., J. Biol. Chem. 267:22624-29, 1992).
CD4+ T cell activation generally follows a multi-step course that includes co-ligation of the TCR and CD4 by the MHC class II/peptide complex presented by APCs. A separate activation event referred to as “co-stimulation” is mediated by other T cell surface molecules, such as CD28. In the absence of the second, co-stimulatory signal, stimulation of T cells through the TCR by MHC class II/peptide complex reportedly induces a state of unresponsiveness to subsequent optimal antigen presentation, commonly referred to as “anergy”. (Quill, J. Immunol 138:3704-12, 1987; Schwartz, J. Exp. Med. 184:1-8, 1996). In other studies, ligation of the TCR in the absence of a costimulatory signal has been reported to disrupt normal T cell activation, inducing a range of responses from anergy to apoptosis (Schwartz, J. Exp. Med. 184:1-8, 1996; Janeway, Cell 76:275-85, 1994; Burrows et al., J. Immunol 167:4386-95, 2001; Wang et al., The Journal of Immunology, 2003).
MHC-restricted T lymphocyte interactions have been widely and extensively investigated. Cells of the T helper/inducer subset generally recognize antigen on the surface of APCs only in association with class II MHC gene products, which results in genetic restriction of antigen recognition. While the rules governing the activation of MHC-restricted T cells, and particularly of class II MHC-restricted T cells, have been well described, the underlying mechanisms are still being defined.
Despite the very large number of possible TCR specificities of T cells, a number of studies have shown that the major portion of the T cell response to protein antigens may be directed to a few “immunodominant” epitopes within the antigenic protein. In the context of autoimmune diseases, class II MHC-restricted T cell responses, and in some cases clinical signs of autoimmune disease, have been demonstrated to be associated with specific proteins and/or immunodominant epitopes from these proteins, including, e.g., type II collagen (Rosloneic et al., J. Immunol. 160:2573-78, 1998; Andersson et al., Proc. Natl. Acad. Sci. USA 95:7574-79, 1998; and Fugger et al., Eur. J. Immunol. 26:928-33, 1996), and human cartilage Ag gp39 (Cope et al., Arthritis Rheum. 42:1497, 1999) associated with rheumatoid arthritis (RA), glutamic acid decarboxylase 65 (Patel et al., Proc. Natl. Acad. Sci. USA 94:8082-87, 1997; Wicker et al., J. Clin. Invest. 98:2597, 1996) and insulin (Congia et al., Proc. Natl. Acad. Sci. USA 95:3833-38, 1998) associated with Type 1 diabetes (insulin dependent diabetes mellitus or IDDM), and myelin oligodendrocyte glycoprotein (MOG) (Forsthuber et al., J. Immunol. 167:7119, 2001) associated with MS and an animal disease model for MS, experimental autoimmune encephalomyelitis (EAE). Similar findings have been reported for class II MHC-restricted T cell responses associated with myelin basic protein (MBP) (Madsen et al., Nat. Genet. 23:343, 1999), proteolipid protein (PLP) (Kawamura et al., J. Clin. Invest. 105:977, 2000), and MOG (Vandenbark et al., J. Immunol. 171:127-33, 2003).
One approach for managing and treating autoimmune diseases and other T cell-mediated immune disorders is to regulate T cell activity using natural or synthetic TCR ligands, or T cell modulatory drugs or other compounds, that are TCR agonists or antagonists. Various analogs of natural TCR ligands have been produced which comprise extracellular domains of class II MHC molecules bound to a specific peptide Ag. Several such constructs have been purified as detergent extracts of lymphocyte membranes or produced as recombinant proteins (Sharma et al., PNAS. 88:11465-69, 1991), Kozono et al., Nature 369:151-54, 1994; Arimilli et al., J. Biol. Chem. 270:971-77, 1995; Nag, PNAS 90:1604-08, 1993; Nag et al., J. Biol. Chem. 271:10413-18, 1996; Rhode et al., J. Immunol. 157:4885-91, 1996; Fremont et al., Science 272:1001, 1996; Sharma et al., Proc. Natl. Acad. Sci. USA 88:11405, 1991; Nicolle et al., J. Clin. Invest. 93:1361, 1994; Spack et al., CNS Drug Rev. 4: 225, 1998).
These two-chain, four-domain molecular complexes loaded with, or covalently bound to, peptide Ag have been reported to interact with T cells and modulate T cell activity in an Ag-specific manner (Matsui et al., Science 254:1788-91, 1991; Nag et al., J. Biol. Chem. 267:22624-29, 1992; Nag, J. Biol. Chem. 268:14360-14366, 1993; Nag, PNAS 90:1604-08, 1993; Nicolle et al., J. Clin. Invest. 93:1361-1369, 1994; Spack et al., J. Autoimmun. 8:787-807, 1995). Various models have been presented for how these complexes may be useful to modulate immune responses in the context of autoimmune disease. For example, U.S. Pat. No. 5,194,425 (Sharma et al.) and U.S. Pat. No. 5,284,935 (Clark et al.) report the use of isolated MHC class II/peptide complexes conjugated to a toxin to eliminate autoreactive T-cells. Others have reported the use of MHC II/antigen complexes, in the absence of co-stimulatory factors, to induce a state of non-responsiveness in Ag-specific T cells known as “anergy” (Quill et al., J. Immunol., 138:3704-3712 (1987). Following this observation, Sharma et al. (U.S. Pat. Nos. 5,468,481 and 5,130,297) and Clarke et al. (U.S. Pat. No. 5,260,422) suggested that soluble MHC II/antigen complexes can be administered therapeutically to anergize T-cell lines that specifically respond to autoantigenic peptides. Additional studies report that soluble MHC II/antigen complexes can inhibit T cell activation, induce T cell anergy, and/or alleviate T cell-mediated symptoms of autoimmune disease (Sharma et al., Proc. Natl. Acad. Sci. USA 88:11405, 1991; Spack et al., CNS Drub Rev. 4: 225, 1998; Steward et al., J. Allerg. Clin. Immun. 2:S117, 1997). In some cases, in the absence of co-stimulation, intact MHC class II/peptide complexes have been reported to modulate T cell activity by inducing antigen-specific apoptosis rather than anergy (Nag et al., J. Biol. Chem. 271:10413-18, 1996).
Although the concept of using isolated MHC/antigen complexes in therapeutic and diagnostic applications holds great promise, a major drawback to the various methods reported to date is that the complexes are large and consequently difficult to produce and work with. While these four domain complexes can be isolated from lymphocytes by detergent extraction, such procedures are inefficient and yield only small amounts of protein. Although cloning of genes encoding MHC complex subunits has facilitated production of large quantities of individual subunits through expression in prokaryotic cells, the assembly of individual subunits into MHC complexes having appropriate conformational structure has proven difficult. Another important feature of these previously described, MHC II/antigen complexes is that they bind not only to the TCR, but also to the CD4 molecule on the T cell surface through the 132 MHC domain (Brogdon et al., J. Immunol. 161:5472, 1998). This additional interaction during peptide presentation and TCR engagement complicates the usefulness of prior MHC II/antigen complexes for certain diagnostic and therapeutic applications. In addition, because of their size and complex structure, prior class II MHC complexes present an inherently difficult in vitro folding challenge.
To overcome these obstacles and provide additional advantages, inventors in the current application previously developed novel, recombinant TCR ligands or “RTLs” for use in modulating T cell activity. These RTLs incorporate selected structural components of a native MHC class II protein, typically comprising MHC class II α1 and β1 domains (or portions of the α1 and β1 domains necessary to form a minimal, Ag-binding pocket/TCR interface). These RTLs may exclude all or part of the 82 domain of the MHC class II protein, typically at least the CD4-binding portion of the β2 domain. Likewise, RTLs for use within the invention may exclude the α2 domain of the MHC class II protein (see, e.g., Burrows et al., Prot Eng. 12:771, 1999). Various RTLs having these general structural characteristics been produced in E. coli, with and without amino-terminal extensions comprising covalently bound, peptide Ag.
These kinds of RTL constructs have been demonstrated to be effective agents for alleviating symptoms of CD4+ T cell-mediated autoimmune disease in an MHC-specific, Ag-specific manner (Burrows et al., J. Immunol 167:4386-95, 2001; Vandenbark et al., Journal of Immunology, 2003). For example, RTL constructs have been tested and shown to prevent and/or treat MBP-induced EAE in Lewis rats (Burrows et al., J. Immunol. 161:5987, 1998; Burrows et al., J. Immunol. 164:6366, 2000) and to inhibit activation and induce IL-10 secretion in human DR2-restricted T cell clones specific for MBP-85-95 or BCR-ABL b3a2 peptide (CABL) (Burrows et al., J. Immunol. 167:4386, 2001; Chang et al., J. Biol. Chem. 276:24170, 2001). Another RTL construct designed by inventors in the current application is a MOG-35-55/DR2 construct (VG312) that potently inhibits autoimmune responses and elicits immunological tolerance to encephalitogenic MOG-35-55 peptide, and alleviates or reverses clinical and histological signs of EAE (Vandenbark et al., J. Immunol. 171:127-33, 2003). Numerous additional RTL constructs useful for modulating T cell immune responses have been developed by the current inventors, which can be effectively employed within the compositions and methods of the instant invention (see, e.g., Huan et al., J. Immunol. 172:4556-4566, 2004).
In recently described protein engineering studies of RTLs, applicants discovered that MHC class II-derived RTL molecules can form undesirable aggregates in solution. In the case of one RTL construct derived from HLA-DR2 (DRB1*1501/DRA*0101)), the purified RTL yielded approximately 10% of the molecules in the form of stable dimers, with a remaining percentage of the molecules found in the form of higher-order structures above 300,000 Daltons (Chang et al., J. Biol. Chem. 276:24170-76, 2001).
Although RTL aggregates retain biological activity (Burrows et al., J. Immunol 167:4386-95, 2001; Vandenbark et al., Journal of Immunology 171:127-133, 2003), conversion of multimeric RTLs into a monodisperse reagents in solution remains an important, unfulfilled objective to facilitate use of RTLs as human therapeutics, for example to treat multiple sclerosis and other autoimmune conditions.
Accordingly, there remains an unmet need in the art to provide recombinant TCR ligands (RTLs) that retain the ability to bind Ag peptides and interface functionally with a TCR to modulate T cell activity in an Ag-specific manner, which have diagnostic and/or therapeutic utility, and which exhibit a reduced potential for aggregation in solution or following administration to a mammalian subject